Wat. Res. Vol. 34, No. 17, pp.

4177±4184, 2000
7 2000 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
PII: S0043-1354(00)00200-1 0043-1354/00/$ - see front matter

www.elsevier.com/locate/watres

DECOLORIZATION OF REACTIVE DYES WITH

DIFFERENT MOLECULAR STRUCTURES UNDER
DIFFERENT ENVIRONMENTAL CONDITIONS
THONGCHAI PANSWAD*M and WORRAWIT LUANGDILOK
Department of Environmental Engineering, Chulalongkorn University, Bangkok, 10330, Thailand

(First received 1 June 1999; accepted in revised form 22 February 2000)

AbstractÐReactive dyes have been identi®ed as problematic compounds in textile wastewaters as they
are water soluble and cannot be easily removed by conventional aerobic biological wastewater
treatment systems. Anaerobic systems could reduce the color intensity more satisfactorily than the
aerobic processes. However, the intermediate products are carcinogenic aromatic amines which need to
be further decomposed by an aerobic treatment. An anaerobic/aerobic SBR system was used to treat a
synthetic dye wastewater with glucose and acetic acid (1000 mg/l COD) as carbon sources, together
with 20 mg/l of four di€erent reactive dyes; i.e., bisazo vinylsulphonyl, anthraquinone vinylsulphonyl,
anthraquinone monochlorotriazinyl and oxazine. The color reduction of the ®rst three dyes was 63, 64
and 66%, respectively. The decolorization eciency of the last or oxazine dye was not determined
because of a strange phenomenon or re-appearance of the color when samples were disturbed. More
color removal was achieved in the anaerobic phase than in the aerobic step. The initial decolorization
rate was 11.9, 0.37 and 0.48 SU/h for the ®rst three dyes, respectively. Though the system comprised
anaerobic and aerobic tanks, the color reduction did not rely on phosphorus accumulating organisms
(PAOs). High temperature and photo-oxidation through exposure to sunlight could increase the
decolorization rate, and the decolorization was not possible if viable organisms were not present in the
system. Nitrate, when present, could interfere with the color reduction while sulfate did not. The bisazo
reactive dye was decolorized by the reductive reaction, which resulted in the cleavage of the azo bond.
Meanwhile, the decolorization of anthraquinone dyes was through the adsorption of dyes on ¯oc
materials. 7 2000 Elsevier Science Ltd. All rights reserved

Key wordsÐcolor removal, reactive dyes, azo dyes, dye wastewaters, biological phosphorus removal,
anaerobic±aerobic process

INTRODUCTION biological phosphorus removal (BPR) system, is

Reactive dyes are very soluble by design and, as a
result, not all are used up by textile ®bers during
the dyeing process and therefore end up with the
discharge from dyehouses (Oxspring et al., 1996).
They are hardly biodegraded under an aerobic en-
vironment (Pagga and Brown, 1986; Randall et al.,
1993). Brown and Laboureur (1983), Carliell et al.
(1995) and Chinwetkitvanich et al. (2000), however,
demonstrated the possibility of decolorization by
anaerobic processes treating azo-reactive dyes. The
aromatic amine intermediates which are produced
under the anaerobic phase are carcinogenic (Baugh-
man and Weber, 1994), but can be further degraded
under the aerobic step to become less harmful end
products (Brown and Hamburger, 1987). An an-
aerobic±aerobic (A±A) process, which can also be a
*Author to whom all correspondence should be addressed.
Tel.: +66-2-218-6669; fax: +66-2-218-6666; e-mail:
pthongch@chula.ac.th
therefore a good choice to simultaneously remove
organics, phosphorus and color. Pansuwan and
Panswad (1997) and Panswad and Iamsamer (2000)
have used the process with moderate to good
results. In this study, an A±A sequencing batch
reactor (SBR) was investigated for its performance
with four di€erent blue reactive dyes. The e€ects of
the nitrate and sulfate as well as other environmen-
tal conditions on the decolorization were also stu-
died.
METHODS AND MATERIALS
This study was divided into two parts. The ®rst part
was performed on a bench scale AA-SBR process and the
latter was performed batchwise using test tubes. The pro-
cess was run at room temperature of about 308C, at an 8
day sludge age (yC), 24 h cyclic time, and with a synthetic
wastewater (Table 1 and Fig. 1). A long anaerobic time
(18 h) was used to maximize the decolorization potential
(Brown and Laboureur, 1983). Details of operation can be

4177
4178 Thongchai Panswad and Worrawit Luangdilok
Table 1. Synthetic wastewater composition
Glucose 860 mg/l
Acetic acid (99.9%) 0.150 ml/l
(NH2)2CO 108 mg/l
KH2PO4 67 mg/l
NaHCO3 840 mg/l
MgSO4
7H20 38 mg/l
CaCl2 21 mg/l
FeCl3
6H20 7 mg/l
Dye 20 mg/l
seen elsewhere (Luangdilok and Panswad, 1999). The
selected four dyes were as follows:
Dye 1 4 Remazol Black B or Reactive Black 5 with
bisazo and sulfate of 2-hydroxyethylslfone structure
Dye 2 4 Remazol Blue R or Reactive Blue 19 with
anthraquinone and sulfate of 2-hydroxyethylslfone
structure
Dye 3 4 Cibacron Blue CR or Reactive Blue 5 with
anthraquinone monochlorotriazinyl structure
Dye 4 4 Procion Blue H-EGN or Reactive Blue 198
with oxazine structure,
of which the chemical structures are shown in Fig. 2. The
initial dye concentration was set at 20 mg/l.
In the second part of the study, which was performed
after the completion of the ®rst part at a steady state,
batchwise experiments in triplicate (three test tubes per
sample) were used to determine the e€ects of ®ve di€erent
environmental conditions, as follows.
1. With or without viable microorganisms when exposed
outdoors: three scenarios were tested, i.e., VO (with
viable organisms), F/St (®ltered and sterilized, i.e.,
samples were GF/C ®ltered and autoclaved for steriliza-
tion and viable organisms were not present), and DC
(dead cells, i.e., non-®ltered samples were autoclaved,
resulting in no viable organisms, but dead cells were
still present).
2. E€ect of photo-oxidation and temperature when viable
organisms were available under three conditions,
namely: RD (refrigerated at 48C in the dark), RC
(room condition, about 308C), and OE (outdoor ex-
posure at 28±388C, with photo-oxidation by sunlight
during daytime).
3. E€ect of temperature: T20 (at 208C), RT (room tem-
perature at about 28±338C), and T40 (at 408C), all in
dark room conditions.
4. E€ect of nitrate: C (control test or no nitrate addition),
N5 and N10 (with 5 and 10 mmol of NOÿ 3 addition, re-
spectively) under a room condition and temperature of
about 308C.
5. E€ect of sulfate: C (control test or no sulfate addition),
Fig. 1. Laboratory set up.
S5 and S10 (with 5 and 10 mmol of SO2ÿ4 addition, re-
spectively) under a room condition and temperature of
about 308C.
It is noted that mixed liquors for the last four conditions
were not pretreated, i.e., neither ®ltered nor autoclaved.
In each test, four to ®ve sets of three test tubes each
(i.e., 3  4 to 3  5 tubes) were used. For each case, 360 to
450 ml aliquots of the mixed liquor were taken from the
SBR tanks 5 min after the dump feeding, pre-treated as
earlier stated for di€erent conditions and ®lled into 12±15
30-ml test tubes. These tubes were left non-aerated and
non-agitated under room temperature, in the dark or with
exposure to sunlight, according to the objective of the
tests shown above. All chemical analyses were done
according to Standard Methods (American Public Health
Association, American Water Works Association and
Water Pollution Control, 1992). Certain samples were ®l-
tered through GF/C paper, while those for soluble phos-
phorus (SP) and color measurements were 0.45 mm ®ltered
before the analysis. The color intensity was determined in
the visible wavelengths of 400±700 nm by a Shimadzu
UV-1201 spectrophotometer, and expressed as space units
(SU) (Gregor, 1992). It is noted that nitrite and nitrate
concentrations were not analyzed due to color interference
from the dyes.
RESULTS AND DISCUSSION
Two carbon sources from glucose (850 mg/l as
COD) and acetic acid (150 mg/l as COD) were
added to the system for co-metabolism, which
could enhance the color reduction eciency (Car-
liell et al., 1995; Razo-Flores et al., 1997; Chinwet-
kitvanich et al., 2000). The process performance
regarding the COD and TKN removal was very
high, i.e., 90±99% (Luangdilok and Panswad,
1999). The color reduction of the azo dye (dye 1)
was about 63% (Table 2). More decolorization
took place in the anaerobic than in the aerobic
stage, i.e., the color dropped from 97.3 in the in¯u-
ent to 37.4 and 36.5 SU in the two steps, respect-
ively.
For the anthraquinone dyes (dyes 2 and 3), the
decolorization performance was 64 or 66%, respect-
ively. For the oxazine dye (dye 4), the decoloriza-
tion was visually obvious in the SBR tank, but
when samples were taken and ®ltered for the color
measurement, re-colorization took place for
unknown reasons (Luangdilok and Panswad, 1999).
The color comparison was therefore not possible.
The data shown in Table 2 and subsequent ®gures
Table 2. Color (SU) reductiona
Dye 20 mg/l dye
Inf. Ana. Aer. %rem.
1 97.3 37.4 36.5 63
2 29.5 12.6 10.5 64
3 43.4 14.1 14.8 66
4 52.6 29.6 31.3 41
a
Average result of 16 days at the steady state, which was achieved
after 40±60 days of prior operation.
 Decolorization of reactive dyes 4179

Fig. 2. Chemical structures of the four selected reactive dyes.

for this oxazine dye were then based on the results
after the re-colorization phenomenon.
The color reduction of the bisazo dye occurred in
the ®rst 2 h of the anaerobic stage (Fig. 3). The in-
itial decolorization rate (IDR) was 11.9 SU/h and
the speci®c decolorization rate (SDR) was 6.72 SU/
g
VSS-h. After that, the DR and SDR dropped to
0.44 SU/h and 0.25 SU/g
VSS-h, respectively. These
latter ®gures are close to those of the two anthra-
quinone dyes, i.e., 0.37, 0.48 SU/h and 0.19, 0.25

Fig. 3. Pro®le of color (SU) of the four dyes vs reaction time.

4180 Thongchai Panswad and Worrawit Luangdilok
Fig. 4. Pro®le of soluble phosphorus (SP) for the four dyes at 20 mg/l concentration.
SU/g
VSS-h, respectively. The decolorization of the
azo dye was due to the reduction and, as a result,
the cleavage of the azo bonds during the anaerobic
step (Carliell et al., 1995; Luangdilok and Panswad,
1999), while that of anthraquinone dyes was from
the adsorption onto bacterial ¯oc materials (Luang-
dilok and Panswad, 1999; Panswad and Techova-
nich, 2000). However, Pagga and Brown (1986)
studied di€erent dyes and also reported the aerobic
decolorization was partly from the adsorption
mechanism. It is noteworthy that essentially no
further color removal took place in the following
aerobic phase. This is in agreement with Gerald
and Bishop (1995), Luangdilok and Panswad (1999)
and Panswad and Iamsamer (2000).
Phosphorus removal
The AA-SBR system for dyes 1 (bisazo) and 4
(oxazine) acted as a BPR process, in which the an-
aerobic phosphorus (P) release and the subsequent
aerobic luxurious P uptake were obvious (Fig. 4).
The phosphorus increased from 16.3 in the in¯uent
to 26 and 28.6 mg/l for dyes 1 and 4, respectively
(Table 3). The phosphorus accumulating organisms
(PAOs) then luxuriously uptook the phosphate in
the aerobic phase, leaving the soluble phosphorus
(SP) at 3.6 and 0.6 mg/l in the e‚uent, respectively.
This occurrence (a better decolorization at a more
inferior P removal for the bisazo dye) demonstrated
that the color reduction in this process did not rely
only on the PAOs. The decolorization could take
place if only the anaerobic step was introduced into
the system. This agrees with Panswad and Iamsa-
mer (2000) and Panswad and Techovanich (2000).
Table 3. Phosphorus removal
Dye 20 mg/l dye
Inf. Ana. Aer.
1 16.3 26 3.6
2 16.3 13.8 7.9
3 16.3 11.7 9.6
4 16.3 28.6 0.6
Viable microorganisms
In the second part of the study, batch test-tube
experiments were used. Figure 5 illustrates the deco-
lorization performance at di€erent scenarios when
test tubes were exposed outdoors for 72 h. In the
control case where samples were not pre-treated
and viable organisms (VO) were present, the deco-
lorization and COD removal rate and eciency
were the highest. No COD removal was seen in the
F/St (®ltered and sterilized) case in which viable
microorganisms or even dead cells were not in exist-
ence, while certain color reduction was still evident.
The decolorization here could not be from the bio-
chemical reactions and must have been through the
photo-oxidation only, and this decolorization ca-
pacity was less than those under the other two con-
ditions. For the DC (dead cells) scenario where no
live organisms but some dead cells were present, the
color reduction was higher than the F/St case, es-
pecially for dyes 1 and 3, but lower than that of
VO condition, even though the COD reduction did
not take place in the test tubes in this DC scenario.
This occurrence clearly showed that the color re-
duction in the DC case was not through the bac-
terial decomposition, even with a co-substrate
availability, but rather via adsorption onto the bac-
terial ¯oc mass in the reactors and the photo-oxi-
dation. This is why the decolorization in the F/St
condition was less than that of DC case, except for
dye 4 where a strange phenomenon of re-coloriza-
tion took place, as earlier mentioned. It is noted
that the COD in the DC case was higher than at t
= 0 h because of the cell hydrolysis during the ster-
ilization or autoclaving process.
E€ect of photo-oxidation and temperature
In this test, samples were not pre-treated and
cells normally existing in the mixed liquor were still
present in test tubes. From Fig. 6, it is seen that the
color reduction for all four dyes was highest under
%rem.
the OE (outdoor exposure) condition and lowest
78 when the samples were refrigerated at 48C in the
52 dark (RD). In this RD condition, due to the inhi-
41
96
bition of bacterial activity at a low temperature, the
COD removal was minimal, and practically no
 Decolorization of reactive dyes 4181

color reduction was achieved. Under room con-
ditions (RC), the COD removal was maximal while
the color reduction was much higher than in the
RD case, but somewhat less than under the OE
scenario. This indicates that the decolorization was
mainly from microbial degradation and to a lesser
degree from photo-oxidation. However, dye 3, the
anthraquinone monochlorotriazinyl dye, seemed to
be more susceptible to the photooxidation than the
other three dyes. In the OE case, a higher decolori-
zation was possible due to two factors, namely, the
photo-oxidation and a higher temperature (28±
388C) during daytime. However, the real appli-
cation of photo-oxidation for decolorization is lim-
ited due to the little exposure to sunlight of normal
treatment tanks. On the other hand, dye waste-
waters are naturally very warm (40±608C) and the
moderately high temperature after some cooling can
be very bene®cial to the system.
E€ect of temperature
All tests regarding the e€ect of temperature were
done in dark rooms and the impact from photo-oxi-
dation was removed. The pro®les of COD and SU
at di€erent times for temperatures of 20, 28±33
(RT) and 408C are shown in Fig. 7. The COD re-
duction at the end of 48 h for the case of room
temperature of 28±338C was the highest, while ap-
proximately the same COD removal was achieved
in the ®rst 24 h. However, more COD than the in-
itial value (at t = 0) was apparent when time pro-
ceeded in some cases. This is because of the same
reason as previously mentioned, i.e., some cells died
and hydrolyzed at elevated temperatures. The color
reduction, however, increased with temperature,
due to higher respiration and substrate metabolism
at the elevated temperatures. This is more obvious
when the results for the azo dye were compared to
those of the anthraquinone dyes. The explanation is
that the decolorization of the azo dye was through
the microbial reaction, which relies on an optimum
temperature, while that of the anthraquinone was
from adsorption onto the ¯oc materials and the
temperature e€ect was not as great.
E€ect of nitrate
All test tubes were placed under a roof at room
conditions in this experiment. The addition of
nitrate could somewhat enhance the COD reduction
rate and eciency (Fig. 8). Denitri®cation by deni-
tri®ers in addition to the anaerobic decomposition
in the tubes was apparently the reason behind this
occurrence. However, more nitrate addition
decreased the azo-dye decolorization capability of

Fig. 5. COD and SU in the experiments on e€ects from viable microorganisms when exposed outdoor
(average from three test tubes per sample): VO (viable organisms at room condition), F/St (GF/C ®l-
tered and autoclaved for sterilization), and DC (non ®ltered samples; dead cells were present after auto-
claving).
4182 Thongchai Panswad and Worrawit Luangdilok

the microorganisms, while it did not have a sub-
stantial e€ect on the color reduction for the anthra-
quinone cases. This is because of the competition in
reduction reaction between the nitrate in the liquid
and the azo bonds in the azo dye, and the nitrate
was obviously a better electron acceptor than the
azo bond. This agrees with the statement by Carliell
et al. (1995). The addition of nitrate did not have
an adverse e€ect on the color reduction for the
anthraquinone dyes because these dyes were
removed by the adsorption mechanisms, not by the
microbial degradation.
E€ect of sulfate
The sulfate e€ect on the COD removal was in the
same pattern as the nitrate impact, i.e., more COD
reduction was evident with a higher sulfate concen-
tration (Fig. 9), possibly because of the action from
the sulfate reducing bacteria (SRB). In other words,
sulfate behaved as an electron acceptor in the pro-
cess in the absence of oxygen. However, the sulfate
as high as 10 mmol did not a€ect the decolorization
capability in the test tubes when the azo dye was
considered, i.e., the same color removal eciency
was achieved regardless of the sulfate concentration.
It may, therefore, be concluded that sulfate is a less
ecient electron acceptor than the azo bond of the
bisazo dye. This is also in agreement with Carliell et
al. (1995). Again, the sulfate addition did not inter-
fere with the color removal for the cases of anthra-
quinone dyes because the decolorization was not
from the microbial activity.
CONCLUSION
The color reduction in the A±A process occurred
mainly in the anaerobic step. Di€erent chemical
structures had certain e€ects on the color and phos-
phorus removal of the system. Viable microorgan-
isms were de®nitely required for a good color
reduction in a biological process, meaning that bio-
logical decolorization is possible. A better decolori-
zation was achieved at elevated temperatures and
when exposed to sunlight, especially for the azo
dye. The photo-oxidation played a minor role in
the decolorization process. Nitrate could interfere
with the color removal of the azo dye because it
was a better electron acceptor, whereas sulfate was
inferior and did not adversely a€ect the decoloriza-
tion capability of the process. The decolorization of
the anthraquinone dyes occurred through the
adsorption on ¯oc materials. Therefore, both nitrate
and sulfate did not have any e€ect on the color
removal eciency. It was not possible to determine

Fig. 6. COD and SU in the experiments on photo-oxidation and temperature e€ects (average from
three test tubes per sample): RD (refrigerated at 48C in the dark), RC (room condition, about 308C),
and OE (outdoor exposure, 28±388C, with the photo-oxidation during daytime).
 Decolorization of reactive dyes 4183

Fig. 7. COD and SU in the experiments on temperature e€ects under dark-room conditions (average
from three test tubes per sample): T20 (at 208C), RT (room temperature, about 28±338C), and T40 (at
408C).

Fig. 8. COD and SU in the experiments on nitrate e€ects (average from three test tubes per sample):
C=control; N5=5 mmol NOÿ 3 ; N10=10 mmol NO3 .
ÿ
4184 Thongchai Panswad and Worrawit Luangdilok
Fig. 9. COD and SU in the experiments on sulfate e€ects (average from three test tubes per sample):
C=control; S5=5 mmol SOÿ2
the decolorization mechanism of the oxazine dye
because of the re-colorization of samples.
AcknowledgementsÐSpecial thanks go to the Thailand
Research Fund, who ®nanced all the experiments in this
study.
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